Wb reagent

The protein lysate (Roche) was added, and the total protein was extracted. Afterward, 50 g of total protein was sampled on a 12% polyacrylamide gel, electrophoresed at 100 V for 2 h, and transferred to polyvinylidene fluoride membranes. After being blocked with 5% skimmed milk at RT for 1 h, the membranes were flushed with TBST three times (10 min each time) and incubated with anti-SIRT1 (ab220807), anti-FOXO3a (ab23683), anti-p-FOXO3a (ab154786), anti-Nrf2 (ab62352), anti-NF-κB (ab220803), anti-p-NF-κB (ab28849), anti-STAT6 (ab32502), and anti-p-STAT6 (ab263947) overnight at 4 °C. All the above antibodies (concentration: 1:1000) were provided by Abcam (Massachusetts, USA). After washing the membranes with TBST, we maintained them with the horseradish peroxidase-tagged antirabbit secondary antibody (1:3000) at RT for 1 h. Next, the membranes were rinsed with TBST times (10 min each). At last, the western blot (WB) reagent (Invitrogen) was applied for color imaging, and the ImageJ 1.44 software was utilized for density assessment and analysis.

After the brain tissues and cells of MCAO rats were treated, we removed the medium. Then, the total proteins were isolated with protein lysates (Beyotime Biotechnology, Shanghai, China). Next, 50 μg of total protein was loaded onto a 12% polyacrylamide gel for 100 V electrophoresis for 2 h and transferred to polyvinylidene fluoride (Millipore, Bedford, MA, USA) membranes. At RT, 5% skim milk was used to block the membranes for an hour. TBST was used to wash them 3 times (10 min each time). Then, the membranes were incubated overnight (4°C) with anti-iNOS (1:1,000, ab178945, Abcam), anti-COX2 (1:1,000, ab179800, Abcam), anti-Bad (1:1,000, ab32445, Abcam), anti-Bax (1:1,000, ab32503, Abcam), anti-Caspase3 (1:1,000, ab13847, Abcam), anti-TREM1 (1:1,000, ab104413, Abcam), anti-TLR4 (1:1,000, ab13556, Abcam), anti-MyD88 (1:1,000, ab133739, Abcam), anti-NF-κB (1:1,000, ab207297, Abcam), anti-p-NF-κB (1:1,000, ab222494, Abcam), and anti-β-actin (1:1,000, ab115777, Abcam). Subsequently, the membranes were rinsed with TBST. The anti-rabbit secondary antibody labeled by horseradish peroxidase (HRP) (concentration: 1:300) was applied for a 1-h incubation at RT. TBST was used to wash the membranes 3 times (10 min/wash). Ultimately, Western blotting (WB) reagent (Invitrogen) was utilized for color imaging. ImageJ 1.44 software was employed for density detection.